Our laboratory investigations have focused on the generation of tumor reactive T cells derived from lymph nodes (LNs) draining sites of progressive tumor or vaccines, and evaluating their anti-tumor reactivity. We have shown that these tumor-primed LNs can be secondarily activated in vitro by anti-CD3 as a surrogate antigen along with other costimulatory molecules (anti-CD28 and anti-4-1BB) to mature into potent effector cells that mediate tumor regression in adoptive immunotherapy. During the last funding period, we found that the in vitro activation of tumor-draining LN (TDLN) cells with an anti-CD3/anti-CD28/anti-4-1BB combination of mAbs resulted in polarization of the cytokine response mediated by the activated cells in response to tumor antigen. Polarized TDLN cells mediated a greater type 1 cytokine (i.e. IFNgamma) response and a decrease type 2 cytokine (ie. IL-4 and IL-10) response compared anti-CD3 activated TDLN cells. Furthermore, the polarized cells manifested significantly enhanced in vivo tumor regression upon adoptive transfer. Also, we have demonstrated that the in vivo administration of anti-4-1BB mAb resulted in the polarization of host lymphoid cells after tumor vaccination. This polarization augmented the therapeutic efficacy of DC-based vaccination in the treatment of established tumors. During the next funding period, we would like to investigate the mechanisms involved with 4-1BB co-stimulation of effector cells; develop additional activation methods to generate tumor-primed lymphoid cells; and, investigate the function of mature effector cells in the lymphopenic host. The specific aims of this proposal are: 1. Examine the in vitro mechanisms of 4-1BB co-stimulation on the polarization of tumor-primed lymphoid cells. 2. Examine the in vivo mechanisms of 4-1BB co-stimulation in T cell immunotherapy. 3. Develop novel methods to activate and expand tumor-primed lymphoid cells. 4. Evaluate the function of adoptively transferred effector cells in the lymphopenic host. The information derived from these studies will provide important insights for the development of immunotherapeutic strategies for the treatment of cancer. We have extensive experience in the conduct of adoptive immunotherapy and vaccine trials in humans that will allow translation of these findings.